Immune Reconstitution Inflammatory Syndrome (IRIS) is the rapid and paradoxical worsening of pathology seen in HIV-1 infected individuals after initiation of anti-retroviral therapy (ART). With the increasing use of ART for mass treatment of AIDS in developing countries, this complication has emerged as a major problem in the management of HIV infection. IRIS is more common in patients with severe CD4 T cell lymphopenia and patients with mycobacterial and other opportunistic infection are at particularly high risk of developing this severe pathologic response. The factors that lead to the induction of IRIS and the mechanisms of its immunopathogenesis are poorly understood, so currently patient treatment during IRIS episodes is limited to supportive care and broad steroid based immunosuppression. We have been engaged in both experimental and clinical studies investigating the mechanisms underlying the immunopathogenesis of IRIS in order to identify patients at risk and to design alternative interventions. As introduced in a previous report we have developed a murine model which recapitulates the key features of the IRIS disease occuring in ART treated HIV patients with mycobacterial co-infections. In this model originated by Dan Barber in the lab we utilized T cell deficient TCR alpha -/- mice infected with M. avium, an important opportunistic pathogen associated with human IRIS. At moderate infectious doses, M. avium infection in T cell deficient mice is asymptomatic for many months despite disseminated bacterial growth. Nevertheless, following adoptive transfer with CD4+ T cells these animals develop a severe wasting disease with impaired lung function and rapidly succumb.In analyzing this form of immune reconstitution disease (IRD), we found that it requires Ag recognition and IFN-gamma production by the donor CD4 T cells, and correlates with marked alterations in blood and tissue CD11b+ myeloid cells. Interestingly, disease was found to be associated with impaired rather than augmented T cell expansion and function, and not strictly dependent on lymphopenia induced T cell proliferation. Instead, our findings (Barber et al., Blood, on line) suggest that mycobacterial-associated IRIS results from a heightened sensitivity of infected lymphopenic hosts to the detrimental effects of Ag-driven CD4 T cell responses. As discussed last year, we have also been engaged in a collaborative clinical study with Drs.Irini Sereti and Mario Roederer aimed at identifying immunologic correlates of IRIS. In this project conducted at the NIH clinical center we characterized T cell phenotypic markers and serum cytokine levels in 45 HIV+ patients with a range of different AIDS defining illnesses, before and at regular time-points after initiation of ART.Patients developing IRIS episodes displayed higher frequencies of effector memory, PD-1+, HLA-DR+ and Ki67+ CD4+ T cells than patients without IRIS. Moreover, PD-1+ CD4+ T cells in IRIS patients expressed increased levels of LAG-3, CTLA-4 and ICOS and had a Th1/Th17 skewed cytokine profile upon polyclonal stimulation. Elevated PD-1 and Ki67 expression was also seen in regulatory T cells of IRIS patients. Furthermore, IRIS patients displayed higher serum IFN-gamma compared to non-IRIS patients near the time of their IRIS events and higher serum IL-7 levels, suggesting that the T cell populations are also exposed to augmented homeostatic signals. These findings (Blood, on line) indicate that IRIS appears to be a predominantly CD4-dependent phenomenon with reconstituting effector and regulatory T cells showing evidence of increased activation from antigenic exposure. Production of type I interferon (IFN) has been linked to the exacerbation of pulmonary tuberculosis (TB) in mice. For example, as shown in a paper published by us this year (Antonelli et al. JCI 2010-see Scientific Advance) TB infected mice treated with the type I IFN inducer PolyIC show increased bacterial loads and pathology. Nevertheless, the role played by type I IFN in human TB is poorly understood. In this study, primary macrophages derived from elutriated monocytes of healthy individuals were infected with MTB or M. bovis Bacillus Calmette-Guerin (BCG), an attenuated vaccine strain, and their responses analyzed by qPCR. We observed that compared to BCG, MTB infection induced markedly lower expression of the NF-kB-dependent pro-inflammatory cytokine genes IL-1 and IL-8 at all time-points examined. On the other hand, transcript levels of the type I IFN associated genes CXCL10 and IFIT1 were significantly higher in MTB infected compared to BCG-infected macrophages. Moreover, neutralization of Type I IFN resulted in decreased growth of MTB and increased proinflammatory cytokine expression in vitro. These findings suggest that the differential regulation of type I IFN is a determinant of mycobacterial virulence in human macrophages and that MTB may exploit the host type I IFN pathway to subvert anti-microbial programs in these host cells.